While our cocktail of antibiotics has been characterized previously as being non- or poorly absorbed from the intestine4272829, we sought to confirm that our behavioral effect was not due to any potential off target effects of the antibiotics. First, to determine if our cocktail of antibiotics was causing the gut to become “leaky” and lead to systemic drug absorption, we measured serum concentrations of vancomycin from animals treated with a prolonged course of oral antibiotics. All 8 of the replicates tested, from a total of 24 antibiotic-treated mice, were fully negative within the limits of this assay (<1.1 μg/ml ± 0). Second, to confirm that the effects of our oral antibiotic treatment were due to local actions within the gut and not due to any unintended systemic effects of the antibiotics, we performed a series of experiments in which mice were injected intraperitoneally with the triple antibiotic cocktail every day – thus bypassing the issue of intestinal absorption. These animals did not show the increased caecal size associated with large-scale knockdown of gut bacteria (data not shown). Since our most
